Supplementary Material for: White Matter Protection with Insulin-Like Growth Factor 1 and Hypothermia Is Not Additive after Severe Reversible Cerebral Ischemia in Term Fetal Sheep

Moderate cerebral hypothermia significantly improves survival without disability from perinatal hypoxia-ischemia. However, protection is partial. Insulin-like growth factor 1 (IGF-1) plays a key role in oligodendrocyte survival and myelination. The purpose of this study was to test the hypothesis that the combination of IGF-1 plus hypothermia could reduce postischemic white matter damage compared with hypothermia alone. Unanesthetized near-term fetal sheep received 30 min of cerebral ischemia, followed by either an infusion of 3 µg of IGF-1 intracerebroventricularly from 4.5 to 5.5 h plus cooling from 5.5 to 72 h (IGF-1 + hypothermia; n = 8), vehicle infusion plus cooling from 5.5 to 72 h (vehicle + hypothermia; n = 12), sham cooling plus sham infusion (ischemia control; n = 12) or sham ischemia (n = 5). The fetal extradural temperature was reduced from 39.4 ± 0.1°C to between 30 and 33°C. White matter was assessed after 5 days. Ischemia was associated with severe loss of CNPase-positive oligodendrocytes in white matter compared with sham ischemia (380 ± 138 vs. 1,180 ± 152 cells/field; mean ± SD; p < 0.001). Delayed hypothermia reduced cell loss (847 ± 297 cells/field, p < 0.01, vs. ischemia control), but there was no significant difference between vehicle + hypothermia and IGF-1 + hypothermia (1,015 ± 211 cells/field; NS). Ischemia was associated with increased caspase 3 expression in white matter (216 ± 41 vs. 19 ± 18 cells/field; p < 0.001). Hypothermia reduced numbers of activated caspase 3-positive cells (116 ± 81 cells/field; p < 0.05), with no significant difference between vehicle + hypothermia and IGF-1 + hypothermia (91 ± 27 cells/field; NS). In conclusion, delayed cotreatment with IGF-1 plus hypothermia after ischemia was associated with an improvement in white matter damage similar to that achieved by hypothermia alone.

亚低温治疗（moderate cerebral hypothermia）可显著改善围产期缺氧缺血（perinatal hypoxia-ischemia）胎羊的无残疾生存率，但其保护作用较为有限。胰岛素样生长因子1（IGF-1）在少突胶质细胞存活与髓鞘形成过程中发挥关键调控作用。本研究旨在验证如下假说：相较于单纯亚低温治疗，联合应用IGF-1与亚低温可更有效地减轻缺血后白质损伤。本研究选用未麻醉的近足月胎羊，先予以30分钟脑缺血造模，随后分为四组接受不同处理：① IGF-1+亚低温组：于造模后4.5~5.5小时脑室内输注3μg IGF-1，并于5.5~72小时实施降温（n=8）；② 溶剂+亚低温组：仅输注溶剂并于5.5~72小时实施降温（n=12）；③ 缺血对照组：假降温+假输注（n=12）；④ 假缺血组（n=5）。实验中，胎羊硬膜外温度从39.4±0.1℃降至30~33℃。于造模后5天对胎羊脑白质进行评估。结果显示，与假缺血组相比，缺血造模可导致脑白质内CNPase阳性少突胶质细胞显著丢失（380±138 vs. 1180±152 个/视野，均值±标准差；P<0.001）。延迟亚低温治疗可减少该类细胞的丢失（847±297个/视野，P<0.01，相较于缺血对照组），但溶剂+亚低温组与IGF-1+亚低温组之间无显著统计学差异（1015±211个/视野；NS）。此外，缺血造模可使脑白质内半胱氨酸天冬氨酸蛋白酶3（caspase-3）的表达水平升高（216±41 vs. 19±18个/视野；P<0.001）。亚低温治疗可降低活化caspase-3阳性细胞的数量（116±81个/视野；P<0.05），且溶剂+亚低温组与IGF-1+亚低温组之间无显著统计学差异（91±27个/视野；NS）。综上，缺血后联合应用IGF-1与延迟亚低温治疗，对脑白质损伤的改善效果与单纯亚低温治疗相当。